WO2020085559A1 - Gène gmpap2.1 dérivé du soja modulant la résistance aux maladies contre le virus de la mosaïque du soja et son utilisation - Google Patents

Gène gmpap2.1 dérivé du soja modulant la résistance aux maladies contre le virus de la mosaïque du soja et son utilisation Download PDF

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WO2020085559A1
WO2020085559A1 PCT/KR2018/013313 KR2018013313W WO2020085559A1 WO 2020085559 A1 WO2020085559 A1 WO 2020085559A1 KR 2018013313 W KR2018013313 W KR 2018013313W WO 2020085559 A1 WO2020085559 A1 WO 2020085559A1
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soybean
plant
gene
protein
mosaic virus
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Korean (ko)
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김국형
위드야사리크리스틴
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서울대학교산학협력단
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8283—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for virus resistance
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/54—Leguminosae or Fabaceae, e.g. soybean, alfalfa or peanut
    • A01H6/542—Glycine max [soybean]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
    • C12Y301/03002—Acid phosphatase (3.1.3.2)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention relates to a GmPAP2.1 gene derived from soybean that modulates disease resistance to soybean mosaic virus and its use.
  • Glycine max is a perennial dicotyledonous plant belonging to the Rosales legumes, known as the country of origin in the northeastern region of Asia, including the Korean Peninsula, and 550 genus 13,000 species are known worldwide, 36 genus 92 in Korea The species is native. Soybeans have been used as a source of protein and fat, and are now in the spotlight as industrial materials.
  • Soybean mosaic virus is a virus belonging to the genus Potyviridae (Potyviridae), which is reportedly transmitted through aphids and up to 43% of seed transmission from soybeans.
  • the virus leads to a decrease in the number of pods directly related to the production of soybeans, a decrease in the number of seeds per pod, a decrease in the size and weight of the seeds, and staining of the bean shell.
  • gene-mediated resistance is one of the defense mechanisms that prevent or reduce viral infection, and plant-resistant genes are classified as recessive and dominant.
  • Recessive resistance genes provide passive resistance that viral proliferation is poorly expressed by incompatible interactions between the virus and the host element, while dominant resistance genes ( R gene) are pathogen effectors or avirulence.
  • R gene dominant resistance genes
  • Avr encoding a resistance protein (R protein) that recognizes the element to provide active resistance.
  • the R gene usually encodes proteins with nucleotide-binding sites (NBS) and leucine-rich repeat (LRR) sites.
  • the NBS domain is composed of functional nucleotide-binding pockets capable of binding and hydrolyzing Adenosine Tri-Phosphate (ATP) and having a signaling resistance response.
  • ATP Adenosine Tri-Phosphate
  • the LRR domain consists of individual iterations of a common leucine-rich repeat motif and has been demonstrated to be related to Avr recognition.
  • This NBS-LRR protein is divided into two groups: the TIR-NBS-LRR protein contains an N-terminal domain with Toll / interleukin-1 receptor homology, and the CC-NBS-LRR protein is N-terminal coiled It features a coiled-coil motif.
  • Rsv3 was identified between the molecular markers A519F / R and M3Satt of the 14th chromosome of the soybean variety L29, and the 5 candidate genes ( Glyma.14g204500, Glyma.14g204600) containing the NBS-LRR domain in the 154 kbp region between the two markers , Glyma.14g204700, Glyma.14g205000 and Glyma.14g205300 ), in particular, it is known that Glyma.14g204700 is closely related to Rsv3 resistance.
  • Rsv4 was identified between the molecular markers Rat2 and S6a of the chromosome 2 of the soybean variety VP-5152, and the NBS-LRR type R gene was not identified, indicating that Rsv4 belongs to a novel class of resistance gene. it means.
  • Korean Patent Registration No. 1552140 discloses a soybean transgenic plant having improved resistance to soybean mosaic virus and a method for manufacturing the same through gene silencing of the HC-Pro gene
  • Korean Patent No. 0857043 Although 'Special Primer for Diagnosing Soybean Mosaic Virus' is disclosed in the issue, the GmPAP2.1 gene derived from soybean that modulates disease resistance to the soybean mosaic virus of the present invention and its use are not described.
  • the present invention was derived by the above-described needs, and the present inventors have the expression of the GmPAP2.1 ( Glycine max purple acid phosphatase 2.1) gene located on chromosome 6 of the soybean variety L29 soybean mosaic virus (SMV) After increasing the infection, it was confirmed that the plant was transformed with a recombinant vector encoding the full-length, N-terminal deletion variant, or C-terminal deletion variant of the GmPAP2.1 protein, and the disease resistance to SMV was analyzed. Transgenic plants expressing the full-length and C-terminal deletion variants of the GmPAP2.1 protein showed high resistance to SMV, while transgenic plants expressing the N-terminal deletion variants of GmPAP2.1 showed reduced resistance to SMV. It was confirmed that the GmPAP2.1 protein confers resistance to soybean plants against SMV, and in particular, by confirming that the N-terminus is an important domain associated with disease resistance to SMV, The present invention has been completed.
  • GmPAP2.1 Glycine max purple acid
  • the present invention transforms plant cells with a recombinant vector containing a gene encoding GmPAP2.1 ( Glycine max purple acid phosphatase 2.1) derived from soybean ( Glycine max ) to encode GmPAP2.1 protein. It provides a method for increasing the disease resistance of the soybean mosaic virus (soybean mosaic virus) of plants compared to non-transformants comprising the step of overexpressing the gene.
  • the present invention comprises the steps of transforming plant cells with a recombinant vector comprising a gene encoding a GmPAP2.1 protein derived from soybeans; And it provides a method for producing a transgenic plant with increased disease resistance to the soybean mosaic virus of the plant compared to the non-transformant comprising the step of re-differentiating plants from the transformed plant cells.
  • the present invention provides a transgenic plant with increased disease resistance to soybean mosaic virus prepared by the above method and a transformed seed thereof.
  • the present invention provides a composition for increasing disease resistance of soybean mosaic virus of a plant comprising a gene encoding a GmPAP2.1 protein derived from soybean consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3 as an active ingredient.
  • soybean-derived GmPAP2.1 gene of the present invention can modulate disease resistance to the soybean mosaic virus, it is expected that it can be applied to increase the productivity of soybeans by developing new varieties with increased resistance to the soybean mosaic virus.
  • Figure 2 is a soybean mosaic virus (soybean mosaic virus, SMV) in G5H or G7H infected soybean plants Gm06g03101 ( GmPAP2.1 ) It is the result of analyzing the expression level of the gene (A), the target protein through sequencing of the Gm06g03101 gene (B), and the position of the Gm06g03101 gene through the chromosome analysis of the soybean variety L29 (C). hpi; Hour post infection.
  • SMV soybean mosaic virus
  • 3 is a phylogenetic analysis of the putative domain of the Gm06g03101 gene.
  • FIG. 4 shows pG5H : GFP, pG7H: GFP, pG5H: GFP- GmPAP2.1 or pG7H: GFP- GmPAP2.1 inoculated into soybean grassy leaves, respectively, to analyze the disease resistance effect of the GmPAP2.1 gene on SMV It is a result of confirming the expression level of GFP by ultraviolet (UV) (A) and a graph (B) showing the expression level of viral RNA.
  • UV ultraviolet
  • pG5H GFP
  • pG5H GFP- GmPAP2.1 (full length)
  • pG5H GFP-GmPAP2.1_N (C-terminal deletion: ⁇ 69-153aa to analyze the disease resistance effect on SMV for each domain of GmPAP2.1 )
  • pG5H GFP-GmPAP2.1_C (N-terminal deletion: ⁇ 1-68aa) was inoculated into the first leaves of soybean respectively, and the expression level of GFP was confirmed by ultraviolet (UV) (A) and the expression level of viral RNA was measured. It is the graph (B) shown.
  • UV ultraviolet
  • the present invention transforms plant cells with a recombinant vector containing a gene encoding a GmPAP2.1 ( Glycine max purple acid phosphatase 2.1) protein derived from soybean ( Glycine max ) to transform the GmPAP2.1 protein. It provides a method for increasing the disease resistance of the soybean mosaic virus (soybean mosaic virus, SMV) of plants compared to non-transformants comprising the step of overexpressing the gene encoding the.
  • SMV soybean mosaic virus
  • the soybean-derived GmPAP2.1 protein may be an N-terminus of the GmPAP2.1 protein or a GmPAP2.1 protein comprising the N-terminus, and preferably may consist of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3, , But is not limited to this.
  • SEQ ID NO: 2 means the entire amino acid sequence of the GmPAP2.1 protein
  • SEQ ID NO: 3 means the amino acid sequence of the N-terminal region of the GmPAP2.1 protein.
  • SEQ ID NO: 3 is an amino acid sequence comprising the 1st to 68th amino acids in the amino acid sequence of SEQ ID NO: 2.
  • GmPAP2.1 protein includes proteins having an amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3 and functional equivalents of the proteins.
  • the term "functional equivalent” as a result of addition, substitution or deletion of an amino acid the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3 at least 70% or more, preferably 80% or more, more preferably
  • the protein has a sequence homology of 90% or more, and more preferably 95% or more, and refers to a protein having substantially the same physiological activity as the protein represented by SEQ ID NO: 2 or SEQ ID NO: 3.
  • “Substantially homogeneous physiological activity” means an activity that increases disease resistance to SMV.
  • the present invention also includes fragments, derivatives and analogs of the GmPAP2.1 protein.
  • fragment refers to a polypeptide having substantially the same biological function or activity as the GmPAP2.1 polypeptide of the present invention.
  • Fragments, derivatives and analogs of the present invention include (i) a polypeptide in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted (the substituted amino acid residues are encoded by a genetic code) May or may not be) or (ii) a polypeptide having substituent (s) at one or more amino acid residues, or (iii) another compound (a compound capable of extending the half-life of the polypeptide, such as polyethylene glycol) and A polypeptide derived from a bound mature polypeptide, or (iv) an additional amino acid sequence (e.g., leader sequence, secretion sequence, sequence used to purify the polypeptide, proteinogen sequence or fusion protein) and It may be a polypeptide derived from the bound polypeptid
  • the gene encoding the GmPAP2.1 protein is characterized by regulating disease resistance to SMV, and the range of the gene includes all genomic DNA, cDNA and synthetic DNA encoding the GmPAP2.1 protein.
  • the gene encoding the GmPAP2.1 protein of the present invention may consist of the nucleotide sequence of SEQ ID NO: 1.
  • homologs of the nucleotide sequence are included within the scope of the present invention.
  • the gene includes a nucleotide sequence having a sequence homology of 70% or more, more preferably 80% or more, more preferably 90% or more, and most preferably 95% or more, respectively, to the nucleotide sequence of SEQ ID NO: 1 can do.
  • % Of sequence homology to a polynucleotide is identified by comparing two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is a reference sequence (not including additions or deletions) to the optimal alignment of the two sequences. Comparison), or additions or deletions (ie, gaps).
  • the term “recombinant” refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a peptide, a heterologous peptide, or a protein encoded by a heterologous nucleic acid.
  • Recombinant cells can express genes or gene segments not found in the natural form of the cells, either in sense or antisense form. Recombinant cells can also express genes found in natural cells, but these genes have been modified and reintroduced into cells by artificial means.
  • vector is used to refer to DNA fragment (s), nucleic acid molecules that are delivered into a cell. Vectors replicate DNA and can be independently reproduced in host cells.
  • carrier is often used interchangeably with “vector”.
  • Vectors of the invention can typically be constructed as vectors for expression or cloning.
  • the vector of the present invention can be constructed using prokaryotic or eukaryotic cells as hosts.
  • a strong promoter for example, pL ⁇ promoter, trp promoter, lac promoter, T7 promoter, tac promoter, etc.
  • a ribosome binding site for initiation of translation and a transcription / detox termination sequence for example, Escherichia coli is used as a host cell, the promoter and operator site of the E. coli tryptophan biosynthetic pathway, and the leftward promoter of phage ⁇ (pL ⁇ promoter) can be used as a regulatory site.
  • the promoters are suitable for transformation, and may be a CaMV 35S promoter, an actin promoter, a ubiquitin promoter, a pEMU promoter, a MAS promoter, or a histone promoter, preferably a CaMV 35S promoter. However, it is not limited thereto.
  • promoter refers to the region of the DNA upstream from the structural gene and refers to a DNA molecule to which RNA polymerase binds to initiate transcription.
  • Plant promoter is a promoter capable of initiating transcription in plant cells.
  • a “constitutive promoter” is a promoter that is active under most environmental conditions and developmental status or cell differentiation. Constitutive promoters may be preferred in the present invention because selection of transformants can be made by various tissues at various stages. Thus, the constitutive promoter does not limit the possibility of selection.
  • the recombinant vector of the present invention can be constructed by methods well known to those skilled in the art. Such methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombinant technology.
  • the DNA sequence can be effectively linked to a suitable promoter in the expression vector to drive mRNA synthesis.
  • the vector may include a ribosome binding site and a transcription terminator as a translation start site.
  • a preferred example of the recombinant vector of the present invention is a Ti-plasmid vector capable of transferring part of itself, the so-called T-region, into plant cells when present in a suitable host such as Agrobacterium tumefaciens .
  • Other types of Ti-plasmid vectors are currently used to transfer hybrid DNA sequences into plant cells, or protoplasts, into which new plants can be produced that properly insert hybrid DNA into the genome of the plant. have.
  • a particularly preferred form of Ti-plasmid vector is the so-called binary vector as claimed in EP 0 120 516 B1 and US Pat. No. 4,940,838.
  • viral vectors such as those that can be derived from double-stranded plant viruses (eg, CaMV) and single-stranded viruses, gemini viruses, etc.
  • CaMV double-stranded plant viruses
  • gemini viruses single-stranded viruses
  • it can be selected from incomplete plant viral vectors.
  • the use of such vectors can be advantageous, particularly when it is difficult to properly transform plant hosts.
  • the recombinant expression vector may preferably contain one or more selectable markers.
  • the marker is a nucleic acid sequence having a property that can be selected by a chemical method, and all genes capable of distinguishing transformed cells from non-transformed cells are included.
  • the marker gene may be an antibiotic resistance gene, but is not limited thereto.
  • a conventional terminator may be used, for example, nopaline synthase (NOS), rice ⁇ -amylase RAmy1 A terminator, phaseoline terminator, Agrobacterium tumefaciens ) Of the Octopine gene, but is not limited thereto.
  • NOS nopaline synthase
  • rice ⁇ -amylase RAmy1 A terminator phaseoline terminator
  • Agrobacterium tumefaciens Of the Octopine gene, but is not limited thereto.
  • terminators it is generally known that such regions increase the certainty and efficiency of transcription in plant cells. Therefore, the use of terminators is highly desirable in the context of the present invention.
  • yeast Saccharomyce cerevisiae
  • insect cells eg, human cells (eg, CHO cell line (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2) , 3T3, RIN and MDCK cell lines) and plant cells.
  • human cells eg, CHO cell line (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2) , 3T3, RIN and MDCK cell lines
  • the host cell is preferably a plant cell.
  • the method for transporting the vector of the present invention into a host cell includes, when the host cell is a prokaryotic cell, a CaCl 2 method, one method (Hanahan, D., 1983 J. Mol. Biol. 166, 557-580) and electroporation. It can be carried out by a method or the like.
  • the vector is injected into the host cell by microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, and gene balm treatment. You can.
  • the present invention also comprises the steps of transforming plant cells with a recombinant vector comprising a gene encoding a GmPAP2.1 protein derived from soybeans; And it provides a method for producing a transgenic plant with increased disease resistance to the soybean mosaic virus of the plant compared to the non-transformant comprising the step of re-differentiating plants from the transformed plant cells.
  • the range of the GmPAP2.1 protein is as described above.
  • Plant transformation refers to any method of transferring DNA to a plant. Such transformation methods do not necessarily have a period of regeneration and / or tissue culture. Transformation of plant species is now common for plant species including both dicotyledonous plants as well as monocotyledonous plants.
  • any transformation method can be used to introduce the hybrid DNA according to the invention into suitable progenitor cells. Methods include calcium / polyethylene glycol method for protoplasts, electroporation of protoplasts, microinjection to plant elements, particle impact methods (DNA or RNA-coated) of various plant elements, infiltration of plants or traits of mature pollen or vesicles Agrobacterium tumefaciens-mediated gene transfer by conversion may be suitably selected from infection with (incomplete) virus and the like.
  • a preferred method according to the invention comprises Agrobacterium mediated DNA delivery.
  • the method for re-differentiating a transformed plant from the transformed plant cell may use any method known in the art.
  • the transformed plant cells must be re-differentiated into whole plants. Techniques for re-differentiation of mature plants from callus or protoplast cultures are well known in the art for numerous different species.
  • the present invention also provides a transgenic plant with increased disease resistance to soybean mosaic virus prepared by the above method and a transformed seed thereof.
  • the plants used in the present invention may be plants belonging to the family Familiar (Family Fabacea), for example, soybean, mung bean, kidney bean, pea, peanut, lentil, eastern, red bean, etc. If the host plant of the soybean mosaic virus, but is not limited thereto.
  • Familiar Familiar
  • the host plant of the soybean mosaic virus but is not limited thereto.
  • the present invention also provides a composition for increasing disease resistance of soybean mosaic virus of a plant comprising a gene encoding a GmPAP2.1 protein derived from soybean consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3 as an active ingredient.
  • the composition comprises a gene encoding the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3 as an active ingredient or a recombinant vector containing the gene, and the gene is transformed into a soybean plant, thereby cultivating the soybean mosaic virus of the soybean plant. It can increase the resistance.
  • Soybean (soybean) varieties L29 (Rsv3) and Somyungkong were pre-sale at the Cultivation Environment Division of the Central Crop Department of the National Institute of Food Science and Technology, and cultured in a growth chamber set at 25 ° C and 16 hours / 8 hours.
  • the L29 variety was used as a target plant for total RNA extraction for cDNA synthesis, and Sombean was used as a target plant for transformation.
  • the seedling of the bean to be inoculated with the viral vector was selected to have the first leaves fully expanded.
  • a coding region (714 bp) of a green fluorescent protein (GFP) without a start codon and a stop codon was amplified.
  • Primer information used in the PCR is shown in Table 1 below, and PCR was performed using Ex Taq polymerase (TaKaRa, Japan) for 35 cycles.
  • PCR amplification products were digested with Mlu I and Xba I restriction enzymes, and then linked to multiple cloning sites (MCS) of viral expression vectors based on SMV-G5H (pG5H) and SMV-G7H (pG7H), and finally produced Was designated G5H: GFP.
  • MCS multiple cloning sites
  • Primer information Primer name Base sequence (5 ' ⁇ 3') (SEQ ID NO) GFP_F 5'-GC TCTAGA GTGAGCAAGGGCGAGGAGCTG-3 '(4) (Italics: restriction enzyme Xba I site) GFP_R 5'-GC ACGCGT GACTGTAAAGATACGGACTC CTTGTACAGCTCGTCCATGCCG-3 '(5) (Italic: restriction enzyme Mlu I site, underline: coding region of NIa protease recognition site)
  • the GmPAP2.1 coding region without termination codon was amplified from cDNA of soybean variety L29.
  • the amplification product was digested with restriction enzyme Mlu I and cloned into pG5H :: GFP, and the final product was named pG5H :: GFP-GmPAP2.1.
  • a mutant clone (pG5H: GFP-GmPAP2.1_N (C-terminal deletion: ⁇ 69-153aa) and pG5H: GFP-GmPAP2.1_C (N-terminal deletion: ⁇ 1-68aa)) was prepared in the same manner as described above. Did.
  • each mutant portion was amplified from plasmid DNA of pG5H :: GFP-GmPAP2.1. Primer information used in the construction of mutant clones is shown in Table 2 below. Insertion of the target gene or fragment of the target gene in the viral vector was confirmed by colony PCR and sequencing.
  • Plasmid DNA was prepared using the Plasmid Maxi kit (QIAGEN, USA), a total of 20 mixed with 10 ⁇ g of plasmid DNA clone and 20X potassium phosphate buffer pH 7.5 (final concentration 1X) in each herbaceous Lub was inoculated with mixed solution.
  • RNA per each leaf was extracted through RNAiSO plus reagent method (TaKaRa), and cDNA was synthesized using GoScript reverse transcriptase (Promega Corp., USA) and oligo (dT) primer (15 mer, Bioneer).
  • Viral RNA replication was performed by amplifying a portion of the coat proteins (CPs) of SMV-G5H. Short fragments of the soybean-derived CDPK (Calcium dependent protein kinase) gene were used as an internal control to standardize other samples, and the information of the SMV-G5H envelope protein fragment and primers for CDPK gene amplification are shown in Table 3 below. same. The experiment was repeated three times.
  • CPs coat proteins
  • Primer information Primer name Base sequence (5 ' ⁇ 3') (SEQ ID NO) SMV-G5H-CP_F 5'-AAGGCTGCAGCTCTCTCGGG-3 '(10) SMV-G5H-CP_R 5'-TCACATCCCT-TGCAGTATGCCTT-3 '(11) CDPK_F 5'-AGTAAAGAGCACCATGCCTATCCAC-3 '(12) CDPK_R 5'-ATGGTTATGTGAGCAGATGCAAGGC-3 '. (13)
  • Gm06g03101 gene of L29 varieties increased during the initial infection of G5H and G7H of SMV, especially after 8 hours of infection, the expression of Gm06g03101 gene increased about 4 times compared to uninfected plants.
  • the Gm06g03101 gene is related to resistance to SMV (FIG. 2A).
  • the nucleotide sequence of the Gm06g03101 gene it was thought to encode the superfamily metalophosphatase purple acid phosphatase-like protein (FIG. 2B).
  • the soybean variety L29 is known to have the Rsv3 gene on chromosome 14, which has strong resistance to SMV-G5H, but the Gm06g03101 gene is located between the Sat130 and BARC_024137_04780 molecular markers of chromosome 6 and is not found near the Rsv3 gene, so it is not found in Rsv3 It was found that it was not related to the gene (Fig. 2C).
  • GmPAP2.1 gene is associated with resistance to SMV
  • pG5H GFP
  • pG7H GFP
  • pG5H GFP- GmPAP2.1
  • pG7H GFP- GmPAP2.1
  • pG5H GFP
  • pG5H GFP- GmPAP2.1
  • full length pG5H
  • GFP-GmPAP2.1_N C-terminal deletion: ⁇ 69-153aa
  • pG5H GFP-GmPAP2.1_C (N-terminal deletion: ⁇ 1-68aa) was inoculated into the soybean lobes of the soybeans, and the degree of virus accumulation was observed through expression of GFP and viral RNA 14 and 21 days after the inoculation.

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  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

La présente invention concerne un gène GmPAP2.1 dérivé du soja et son utilisation pour moduler la résistance aux maladies contre le virus de la mosaïque du soja. Le gène GmPAP2.1 dérivé du soja, de la présente invention, peut moduler la résistance aux maladies contre le virus de la mosaïque du soja. Par conséquent, d'après les prévisions, la présente invention peut être appliquée pour développer de nouvelles variétés présentant une résistance accrue au virus de la mosaïque du soja afin d'augmenter ainsi la productivité du soja.
PCT/KR2018/013313 2018-10-24 2018-11-05 Gène gmpap2.1 dérivé du soja modulant la résistance aux maladies contre le virus de la mosaïque du soja et son utilisation WO2020085559A1 (fr)

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KR1020180127200A KR101964658B1 (ko) 2018-10-24 2018-10-24 콩 모자이크 바이러스에 대한 병 저항성을 조절하는 콩 유래의 GmPAP2.1 유전자 및 이의 용도

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